One of the major dangers of living and working in a highly complex and technologically advanced society is that we are exposed to a sea of hazardous or toxic substances that have contaminated our living and working environments, the overwhelming majority of which are man-made and often carcinogenic. According to a recent estimate, there are presently over 70,000 chemicals now in commercial production and another 700-3,000 new chemicals are being introduced every year. Among this vast number of industrial chemicals, only approximately 10% have been tested for toxicity, usually in the form of carcinogenicity, in life-time animal studies--in many cases only after extensive or prolonged human exposure. These statistics do not include the profound changes now occuring as a result of exposure to pollutants in water and air which have secondary or tertiary effects upon the bioatmosphere and the natural biological cycles whose importance we are only now recognizing. With so many untested industrial chemical compounds and chemical changes constantly occuring, it has become logistically impossible to test even a significant portion of them for toxicity (in the form of carcinogenicity) by classical toxicological protocols using animals primarily because there is a lack of manpower, insufficient facilities, and inadequate time and financial resources available to undertake this task on such a mammoth scale. This has led directly to the development of in vitro assay methods which are short-term test systems and involve a minimum of manpower and supplies.
A fundamental problem, which has developed as our knowledge has advanced and the variety of test methodologies and systems has expanded and multiplied, has been a lack of precision and consistency in terminology. In view of the unfortunate tendency to use one term for a variety of meanings or applications and in view of the creation of novel terms which are not precisely defined or understood, the following definitions and terms are provided which will aid to clarify and particularly point out major differences and distinctions.
Toxicity and cytotoxicity: specifically induced cellular injury, usually chemically, which occurs in a sequence of phases which may be almost instantaneous or occur as a progressive series over time. The severity of the injury in the cell may be so extreme that the cells are killed almost instantaneously; alternately, the injury will be more subtle, time consuming and be observed as a series of degenerative changes prior to cell death. For all substances other than very rapidly acting agents, the events are considered as occuring in two phases; the early reversible changes and the late irreversible cell changes ending in cell death. Note in particular that carcinogenicity does not necessarily correlate with toxicity; in fact, there are many cytotoxic non-carcinogens and noncytotoxic carcinogens. Toxicity, by definition therfore, includes within it the terms "carcinogenic" and "degenerative".
Carcinogens (or carcinogenic agents): substances for which there is conclusive evidence from human studies which indicates there is a direct causal relationship between exposure to the substance and human cancer. These are to be distinguished from substances which may be "reasonably anticipated to be carcinogens" which are defined and distinguished as those for which there is only limited evidence of a direct causal relationship between exposure and human cancer or sufficient evidence of such a causal relationship in experimental animals. The term is thus limited to situations where cancer or tumor cells (and sarcomas) arise in humans and other animals after exposure without reference to an action mechanism and thus is a functional, operational definition limited to cellular alteration which results in an observable loss of control of normal cell growth leading to unrestrained proliferation and the formation of tumor.
Degenerative substances: compounds inducing cell injury which does not result in the formation of a tumor or cancer cell as such. The degeneration of the intact cell may in fact be reversible after the initial injury; such reversible changes occur early in time and include mild cytoplasmic edema, dilation of the endoplasmic reticulum, slight mitochondrial swelling, disaggregation of polysomes and the occurrence of small aggregates of chromatin around the nucleus. The irreversible changes occur late in the sequence of progressive steps and include expensive mitochondrial swelling with cristae disruption, gross cytoplasmic swelling with dissolution of organelles, plasma membrane rupture, and nuclear dissolution. In vivo clinical/diagnostic observation and examination of human and animal subjects who have been exposed to degenerative substances typically exhibit emphysema, kidney and liver dysfunction, abnormal neurological function, "wasting syndrome"--a loss of body weight and appetite, and other debilatating states The degenerative effects will vary in severity with the dose of exposure in concentrated or dilute form, whether short or prolonged in duration, and in nature with the wide variety of cellular destruction and target areas within the cell which can eventually lead to cell death. This term, therefore, is an operational, functional definition used to identify all types of cell injury regardless of target area or action mechanism which do not give rise to tumor cells as such.
Genotoxicity (and genotoxic substances): a mechanism of induced cell injury which is limited to direct alterations and modifications of the genetic material in the cell exclusively and include DNA damage, gene mutation, and chromosonal effects in both microorganisms (bacteria and fungi) and mammalian cell systems. The mutations are detected as phenotypic changes and can result from alterations in the structure of DNA as base substitutions, frame shifts, large deletions, insertions, and translocations. The DNA damage is measured either by strand breakage or fragmentation or is measured indirectly as consequent DNA excision and repair. The chromosomal effects include multilocus chromosome deletions and non-disjunction as well as chromosomal aberrations such as breaks, terminal and interstitial deletions, rings, translocations, and dicentrics. A common effect of such modifications of genetic material within mammalian cells is the morphological transformation of the normal cell as altered colonies or foci in a monolayer of cells generally characterized by the piling up of the cells in an irregular, criss-cross pattern representing a loss of growth inhibition in cell-to-cell orientation.
Epigeneticity (and epigenetic substances): a mechanism of induced cellular injury which does not involve direct modifications of genetic material. The effected cells may be destroyed progressively over time or be killed almost instantaneously. The epigenetic substance may cause the cell to become a tumor cell and thus be a "carcinogenic" agent or cause progressively debilitating effects in the cell and thus be termed a "degenerative" substance. The term is therefore an operational and functional definition which identifies an action mechanism by which those substances and agents which are non-genotoxic injure the cell in non-genetic pathways.
Tumor initiator: A chemical compound capable of initiating or inducing cell change and injury in normal cells and rendering them into a premalignant state. Although tumor initiators are generally genotoxic agents, this term definitionally is not restricted to this action mechanism.
Tumor promoter: A substance capable of increasing the incidence of tumor formation in vivo when it is applied repeatedly to an animal after it has previously received an appropriate dose of a tumor initiator. A tumor promoter by itself cannot induce tumor formation nor can it initiate alterations in normal cells which result in the formation of premalignant cells. This term (and its counterpart, "tumor initiators") are also applicable to cultured cells which have been morphologically transformed in vitro into tumor or malignant cells. This term, by definition, does not make any indication or reference to the precise nature or action mechanism of cell injury leading to the transformation. In contrast, substances or agents which act as both initiators and promoters themselves are said to be "carcinogenic" and are not the equivalent of either a "tumor initiator" or a "tumor promoter" as such.
Cytoskeleton pertubation: The disruption or disorganization of specific cytoplasmic filamentous elements or components within the cell without reference to or specification of a mechanism of cellular alteration. The disruptions comprise the disassembly or the depolymerization of microtubules, intermediate filaments and microfilaments, elements which collectively identify and form the complex network of organized filamentous structures within the cell proper. The distribution and organization of these elements in the cytoplasm, although varying from cell type to cell type, is substantively involved in regulating the cellular morphology and shape, motility, mobility of surface receptors, and internal organization of the cell. Any significant change in the structure or organization of these cytoskeleton elements results in a microscopically visible manifestation of cellular injury within the cell.
The technical advances leading to the development of short-term test systems for the detection of toxic or hazardous substances, particularly carcinogenic agents, has grown enormously in recent years. Much detailed information regarding toxicity testing using bacterial and mammalian cell assays and their limitations is found in the following publications: Cellular Systems For Toxicity Testing (Williams et al, editors)Annals Of The New York Academy Of Sciences, Volume 407, 1983; Third Annual Report On Carcinogens Summary, September 1983, Public Health Services, U.S. Department of Health and Human Services; National Toxicology Program, Ad Hoc Panel Draft Report On Chemical Carcinogenesis Testing and Evaluation, 1984, U.S. Gov't. and National Toxicology Program Annual Plan, 1983. In view of these publications and the many references cited therein, the overall effect and state of test systems for the detection of toxic substances may be summarized as follows. Although long-term animal studies have been traditionally accepted as the only experimental laboratory method suitable for providing conclusive evidence of either toxicity or carcinogencity of a substance, short-term in vitro test methods are available which are more efficient, economic and faster. Although many different systems have been proposed for such testing purposes [reviewed extensively by Hollstein et al., Mutat. Res. 65:133-226 (1979)], only a relative few have been recognized as being accurate and have thus correspondingly received significant widespread use including incorporation into the genetic toxicity testing program under present evaluation by the National Toxicology Program. These include: the Salmonella/Ames assay [Ames et al., Mutat. Res. 31:347-364 (1975) and subsequent modifications; the mouse lymphoma systems for point mutations [Amacher et al., Mutat. Res. 64:391-406 (1979)]; the CHO system for chromosome aberrations and sister chromatid exchange (SCE) [Evans, Ann. N.Y. Acad. Sci. 407:131-142 (1983); Wolff, Ann. N.Y. Acad. Sci. 407:142-153 (1983)]; and the Drosophila mutagenesis assay [Rasmuson et al., Mutat. Res. 54:33-38 (1978); Vogel et al., in The Predictive Value of Short Term Screening Tests in Carcinogenecity (Williams et al.,editors), Elsevier/North Holland Biomedical Press, Amsterdam, The Netherlands, pages 125-147, 1980]. Other tests under present evaluation include assays for unscheduled DNA synthesis, aneuploidy, chromosome aberrations and SCE in vivo, cell transformation, and cell to cell communication (metabolic cooperation) [Williams et al., Ann. N.Y. Acad. Sci. 407 (1983); The Use of Human Cells For The Evaluation of Risks From Physical and Chemical Agents (Castellani, ed.), Plenum Press, N.Y. 1983; Chemical Mutagens Principles and Methods For Their Detection (de Serres and Hollaender, eds.), Volume 8, Plenum Press, N.Y. 1983].
The common feature of all these short-term in vitro assay systems is their reliance and dependence upon genotoxic mechanisms per se. By definition therefore, all these assay methods identify the presence of a toxic substance that directly damages DNA or causes cellular injury by inducing alterations and/or modifications in the genetic material as measured by mutagenesis or chromosomal effects directly or indirectly. This limitation and restriction has been deemed to be both acceptable and reliable because there is a very high correlation between genotoxicity per se (and genotoxic test systems by extension) and carcinogenicity (evidence of cancers in humans or animals clinically identified). Consequently, genotoxicity in several test systems has been taken as presumptive evidence of carcinogenicity [Williams and Weisburger, Ann. Rev. Pharm. Tox. 21:393-416 (1981); Weisburger and Williams, Science 214:401-407 (1981); Ames and McCann, Cancer Res. 41: 4192-4203 (1981); Brusick, Ann. N.Y. Acad. Sci. 407:164-176 (1983); Bartsch, Ann. N.Y. Acad. Sci. 407:351-361 (1983)]. Comparison of the data revealed in these publications and many others show that certain classes of carcinogens (e.g., polycyclic aromatic hydrocarbons, alkylating agents, nitrosamines, aromatic amines and others) uniformly showed an excellent (90-100%) genotoxicity (mutagenicity)--carcinogenicity correlation. However, other classes of recognized carcinogens, best exemplified by certain metals, steroid hormones and chlorinated hydrocarbons as found in pesticides and tumor promoters consistently showed very poor responses in the Ames and/or mammalian cell genotoxic assay systems. This has led to the recognition that carcinogens in general should be classified operationally as genotoxic and epigenetic agents [Williams, Ann. N.Y. Acad. Sci., 407:328-333 (1983) and the references cited therein]. By this proposal, those substances, particularly carcinogens, which are capable of interacting directly with genetic materials such as DNA are termed genotoxic exclusively and can be identified by mutagenicity tests currently in use; on the other hand, epigenetic substances including not only carcinogens but also tumor promoters and other toxicants, do not induce cellular damage leading to observable genetic alterations or mutations and in fact can be identified only poorly by the presently available short-term tests. Although the need for non-genotoxic short-term reliable tests for the detection of epigenetic substances has now been recognized, no validated in vitro tests for this purpose are yet available. A major gap in all short-term testing programs thus exists in all investigations and studies now employed for the detection of toxic substances generally.